Butanol can be used as a chemical intermediate in cosmetics, perfume, hormone, a sanitizer, an industrial coating agent, a paint additive, fiber, a plastic monomer, medical supplies, vitamins, antibiotics, pesticides, or the like (Document [Durre, Biotechnol. J, 2:1525-1534, 2007]).
As the existing method for preparing butanol, a method of fermenting sugar using Clostridium strain to produce butanol, acetone, and ethanol (Document [U.S. Pat. No. 1,315,585]) has been used until the 1980s, but thereafter, an oxo process of synthesizing butanol from propylene obtained from petroleum has been widely used. However, since the method for preparing butanol based on petroleum is complicated due to using high temperature and high pressure and large amounts of hazardous wastes and carbon dioxide are discharged (Document [Tsuchida et al., Ind. Eng. Chem. Res., 45: 8634, 2006]). Recently, a demand for eco-friendly producing butanol from sustainable resources through microbial fermentation has been increased again.
However, as described above, currently, in most cases, butanol has been produced by a chemical synthesis method. An interest in research into biobutanol has been rapidly increased around the world due to an increase in oil price, environmental problems, and the like, but biobutanol has not yet been efficiently produced.
In the case of butanol, up to now, in most of the examples of producing butanol through fermentation, Clostridium strain is used. There is an example in which productivities of acetone, butanol, and ethanol were increased by 95%, 37%, and 90%, respectively, as compared to wild-type strains by inserting three genes, that is, acetoacetic acid decarboxylase (adc), CoA transferase A (ctfA), and CoA transferase B (cftB), into a vector and using a promoter of adc to thereby construct an artificial operon and then introducing this plasmid pFNK6 into Clostridium acetobutylicum ATCC 824 strains (Document [Mermelstein et al., Biotechnol. Bioeng., 42:1053, 1993]). In addition, there is an example in which the recombinant strain cloning and expressing alcohol/aldehyde dehydrogenase(aad) produced higher amount of butanol and ethanol than aceton when compared with wild type (Document [Nair et al., J. Bacteriol., 176:871, 1994]). Otherwise, as a method for inactivating a function of genes, there is an example of inactivating butyrate kinase (buk) and phosphotransacetylase (pta). It was reported that when a strain PJC4BK in which the buk gene was inactivated was fermented at pH of 5.0 or more, a production amount of butanol was significantly increased up to 16.7 g/L (Document [Harris et al., Biotechnol. Bioeng., 67:1, 2000]). However, it was reported that when the case of a strain in which a pta gene was inactivated was compared with the case of a wild-type strain, there was no significant difference in producing a solvent (Document [Harris et al., Biotechnol. Bioeng., 67:1, 2000]). In addition, it was reported that when fermentation was performed using a Clostridium beijerinckii BA101 strain, which is a mutant strain induced by random mutation, and maltodextrin as a carbon source, 18.6 g/L of butanol was produced (Document [Ezeji et al., Appl. Microbiol. Biotechnol., 63:653, 2004]). However, even in the case of using these recombinant strains, the production amount of butanol in a culture medium was significantly low (20 g/L or less) due to toxicity of butanol, which is a final product, such that it was impossible to industrially use these recombinant strains. Therefore, various method for extracting butanol in situ produced during a culture process to maintain a concentration of butanol in a culture medium at a level at which cytotoxicity is not generated have been developed. For example, it was reported that productivity may be increased by adsorbing butanol produced during continuous culture using activated carbon (Document [U.S. Pat. No. 4,520,104]).
However, in this method, only butanol is selectively adsorbed by the activated carbon and a concentration of the adsorbed butanol is low, such that it is difficult to recover butanol, and physical stability of activated carbon is insufficient, such that it is impossible to reuse the activated carbon. Therefore, there is a disadvantage in that extraction of butanol is expensive. An adsorption amount of butanol is in proportion to a concentration of butanol, but the concentration of butanol produced in continuous culture is low, such that the adsorption amount of butanol is also significantly low. Due to this problem, in spite of the continuous culture process, the productivity is not over 1 g/L/h. In addition, a column filled with the activated carbon may be physically clogged due to aggregation of cells, thereby causing a problem in a process. In this method, cell aggregates may clog the column and form a channel in a flow of the culture medium, such that it is difficult to allow the products such as butanol, acetone, isopropanol, ethanol, or the like, to be adsorbed in the entire adsorbent, thereby decreasing adsorption efficiency. A method of using an adsorbent except for activated carbon to increase productivity and a concentration of a solvent and using a recyclable adsorbent has been reported (Document [Nielsen et al., Bioeng. Biotech. 102:811-821, 2009]). The method is a method of adding the adsorbent in a culture medium and adsorbing butanol produced during a culture process in the adsorbent to recover butanol. According to this method, the adsorbent should be recovered from the culture medium, and essentially, a loss of the adsorbent is generated in a recovery process. In addition, impurities produced by microbes in the culture process and sugar, which is a raw material, are simultaneously adsorbed, such that purity and productivity of butanol are low at the time of recovering butanol. Further, an adsorption amount of butanol is in proportion to an amount of adsorbent added to the culture medium, but in this method, there is a limitation in an addition amount of the adsorbent in the culture medium. In addition, in this method, concentrations of ethanol and acetone are relatively high, which serves to desorb the adsorbed butanol, such that there is a limitation in increasing the concentration of butanol.